Abstract

Objectives The aims of this study were to investigate the feasibility of contrast-enhanced ultrasound (CEU) imaging for in vivo visualization of intraplaque neovascularization and to correlate the in vivo observations with histological assessment of neovessel density and plaque composition in an experimental animal model of advanced atherosclerosis.

Background Recent evidence has linked plaque angiogenesis with enhanced atherosclerotic plaque progression and vulnerability. Increased neovascularization has been detected in ruptured human lesions and is associated with clinical manifestations of plaque rupture.

Methods Advanced aortic atherosclerosis was induced in New Zealand white rabbits (n = 21; high cholesterol–rich diet/double-balloon aortic denudation). Animals underwent standard and CEU imaging at the end of the atherosclerosis induction period. Six age-matched animals served as control subjects. Within 24 h, animals were euthanized and aortas processed for histopathological evaluation of plaque composition and neovascularization. Imaged plaques were classified as contrast enhanced (CE) positive or CE negative, according to their contrast enhancement on CEU imaging. The lesions were also classified as class III (predominantly echogenic) or class II (predominantly echolucent), according to their echogenicity on non-CEU images.

Conclusions CEU imaging is a feasible noninvasive imaging modality to evaluate intraplaque neovascularization. A noninvasive imaging modality to assess lesion neovascularization could be of great importance to identify vascularized, “high-risk” lesions before rupture.

Cardiovascular events, resulting from plaque rupture and occlusive atherothrombosis, are still the number one cause of death in the Western countries, with approximately 1.2 million heart attacks and 750,000 strokes afflicting the American population each year (1). Accumulating evidence has linked plaque angiogenesis with enhanced lesion progression and vulnerability (2,3). Increased neovascularization has been detected in ruptured human aortic plaques and is frequently associated with pathological signs of plaque vulnerability such as intraplaque hemorrhage and thin-cap fibroatheromas (4–9). Therefore, the availability of a noninvasive technique capable of reliably identifying lesion neovascularization could be particularly attractive for early detection of high-risk, prone-to-rupture plaques before the occurrence of clinical events.

Contrast-enhanced ultrasound (CEU) imaging has been proposed as a feasible and effective technique to detect neovascularization of human atherosclerotic plaque (10–12). The direct visualization of contrast microbubbles within plaque neovascularization was previously described (13,14). These findings were confirmed by a more recent study correlating CEU imaging of carotid arteries with the presence and degree of intraplaque neovascularization as corroborated by histology of the same lesions (15). Visualization of adventitial vasa vasorum in carotid arteries in patients with or without carotid atherosclerosis was recently reported using CEU imaging (16). Despite the promising potential application of CEU imaging in the clinical setting, few studies have included histological validation of the imaging results. The ones that did were limited to high-risk lesions collected from patients undergoing surgical procedures. Moreover, the fact that carotid endarterectomy specimens do not include the whole arterial wall limits the histological evaluation to intraplaque neovascularization and may exclude the assessment of adventitial vasa vasorum.

The aims of this study were to investigate the feasibility of CEU imaging for in vivo visualization of intraplaque and adventitial neovascularization in an experimental animal model of advanced atherosclerosis and to correlate the in vivo observations with a systematic histological assessment of neovessel density and plaque composition.

Methods

Experimental design

Aortic atherosclerosis was induced in New Zealand white male rabbits (n = 21) by a combination of 0.2% cholesterol-rich diet and aortic balloon endothelial injury. Six age-matched animals maintained on normal chow served as control subjects. At the end of the atherosclerosis induction, all animals underwent standard and CEU imaging of the aorta, using a crossover design. The animals were randomized to initial ultrasound imaging with either sonographic contrast or saline, and after an interval of 48 h, the studies were repeated using the other agent (saline/contrast). On completion of the imaging studies, the rabbits were euthanized and their aortas processed for histopathology. The study protocol was approved by the Institutional Animal Care and Use Committee.

Atherosclerosis induction

Atherosclerosis was induced using a previously described protocol (17,18). Briefly, male New Zealand white rabbits (3.9 ± 0.5 kg) were fed a 0.2% cholesterol atherogenic diet (Research Diets Inc., New Brunswick, New Jersey) for 9 months. At 12 and 24 weeks of atherogenic diet initiation, aortic balloon endothelial denuditions were performed with a 3- to 4-F Fogarty catheter (Edwards Lifesciences, Irvine, California) under fluoroscopic guidance. The femoral artery was carefully dissected to avoid any nerve damage, and the catheter was progressed until the thoracic descending aorta. The balloon was gently inflated and pulled back until the iliac bifurcation. This procedure was repeated 3 times. Subsequently, the catheter was removed and the femoral artery closed. Anesthesia for the aortic denuditions and imaging process was induced by intramuscular injection of ketamine (30 mg/kg) and xylazine (2.2 mg/kg). Imaging studies were performed as described in the following ultrasound imaging section. After completion of the imaging studies, animals were euthanized by pentobarbital overdose (75 mg/kg, Sleepaway, Fort Dodge, Fort Dodge, Iowa), and the aortas were harvested and processed for histopathology. All animals received humane care in compliance with the Guidelines for the Care and Use of Laboratory Animals.

Ultrasound imaging

Ultrasound imaging was performed (Philips iEE33 ultrasound machine, Philips, Bothell, Washington), using a 15-MHz linear probe. Image sequences were not electrocardiography gated and the second harmonic detection technique was used for signal detection. First, the infrarenal abdominal aorta was imaged and the plaques localized at the far (posterior) wall of the vessel were selected. This anatomic landmark allowed for a more accurate identification of the selected plaques in the second ultrasound examination as well as for histopathology, using renal arteries as anatomic landmarks. This criterion was not a limitation in identifying atherosclerotic plaques because all the atherosclerotic rabbits showed plaques at the infrarenal tract. Each plaque was classified according to its echogenicity during ultrasound imaging, using a conventional classification scheme reported previously (15): class I = uniformly echolucent; class II = predominantly echolucent; class III = predominantly echogenic; class IV = uniformly echogenic; and class V = extensive calcification with acoustic shadows.

After standard ultrasound imaging, animals underwent CEU imaging of previously identified lesions using saline or contrast agent. The sonographic contrast agent perflutren lipid microspheres (Definity, Bristol-Myers Squibb Medical Imaging, North Billerica, Massachusetts) was intravenously injected (0.2 ml containing 2.4 × 109 perflutren lipid microspheres) via the marginal ear vein and flushed with 1 ml of saline. Image settings were adjusted to maximize contrast signal visualization and a low mechanical index was used (0.06 to 0.08). A preliminary study was performed to establish the most favorable dose and administration as well as optimization of image settings. The studies were digitally stored for subsequent analysis. Standard and contrast-enhanced images were reviewed offline by 2 readers (E.B., F.F.).

During CEU examinations, plaques appeared dark and hypoechoic because of tissue signal suppression. The movement of the echogenic bubbles into the previously identified atherosclerotic lesions generated moving bright spots within the adventitia and the core of the plaque. According to plaque contrast-agent enhancement, each lesion was categorized either as CE negative (no signal within the plaque or confined to plaque adventitial side) or CE positive (signal reaching the plaque core and/or extensive contrast agent enhancement throughout the lesion), as previously described (15).

Contrast signal enhancement was quantified by a customized semiautomatic analysis program (MATLAB, The MathWorks, Inc., Natick, Massachusetts). The regions of interest, including plaque and adventitia, were set in the ultrasound B-mode images, in a sequence of 10 images before and after contrast/saline injection. Mean gray scale of the images pre- (baseline) and post-injection was calculated and contrast-enhancement expressed as the percentage of increase of mean gray scale versus baseline (pre-infusion) image. Interobserver variability data for the lesion classification was 5% and for the quantitative data was 6%.

Histopathology and immunohistochemistry

After the final imaging session, the animals were euthanized and the abdominal aorta perfused with phosphate-buffered saline 1× and fixed in 4% paraformaldehyde in phosphate-buffered saline as previously described (17,19). Briefly, the infrarenal aortic tract that was analyzed by CEU imaging was cut into 3-mm thick cross sections and embedded in paraffin. Serial sections (5-μm thick) were obtained and stained with combined Masson elastin stain and hematoxylin and eosin. Additional serial sections were assessed for macrophage and smooth muscle cell density by using specific antibodies against RAM-11 (1:100 dilution, Dako, Carpenteria, California) and α-actin (1:100 dilution, Sigma-Aldrich, St. Louis, Missouri), respectively. Isolectin B4 (1:100 dilution, Vector Laboratories, Burlingame, California), staining was performed for characterization and quantification of neovessels. Negative controls were obtained omitting the primary antibodies.

Planimetric analysis of the cellular composition of the lesions was performed using a computer-based quantitative color image analysis system (Image-Pro Plus, MediaCybernetics, Bethesda, Maryland) to assess the percentage of the RAM-11– and α-actin–stained area for each section. The macrophage and smooth muscle cell density of atherosclerotic plaques were quantified and expressed as the percentage of plaque area. The number of neovessels was counted and related to the cross-sectional plaque area. Neovessel density (n/mm2) was used for correlation with imaging data.

Statistical analyses

Data are presented as mean ± SD or as median with minimum and maximum if not normally distributed. Test of normality was performed using Kolmogorov-Smirnov and Shapiro-Wilk tests. One-way analysis of variance was used to compare differences between groups. The Kruskal-Wallis test was used as appropriate to compare differences between groups for not normally distributed variables (contrast enhancement at computerized analysis, RAM-11). Pearson correlation was applied for linear association between contrast enhancement and intraplaque neovascularization.

Results

CEU imaging

Atherosclerotic lesions at the infrarenal tract of aorta were detected in all the animals using standard ultrasound examination. A total of 21 lesions were identified. Eight plaques (38%) were classified as class II (predominantly echolucent), whereas the remaining 13 lesions (62%) were categorized as class III (predominantly echogenic) (15). No atherosclerotic plaques were detected in the control unmanipulated animals.

After administration of the contrast agent, the aortic lumen appeared opaque for approximately 1 min in both normal and atherosclerotic rabbits. Atherosclerotic lesions, dark and hypoechoic before contrast agent injection, became bright and visible 30 to 60 s later due to the penetration of contrast within the lesion and remained visible for up to 5 min. Observed contrast enhancement was represented by moving bright spots within the plaque (Online Video 1). In contrast, no signal enhancement was evident in control (nonatherosclerotic) animals. No lesions were apparent after saline administration in atherosclerotic animals. Images obtained with CEU were used to classify lesions into CE positive (53%) or CE negative (47%) by visual examination. CE-positive lesions showed a greater plaque enhancement when evaluated by the semiautomated computerized analysis compared with CE-negative plaques (Fig. 1A). Lesions classified as class III (predominantly echogenic) displayed a significantly higher contrast enhancement compared with class II or predominantly echolucent lesions (Fig. 1B). No contrast enhancement, by either visual or computerized analysis, was observed in control animals.

(A) Plaque area was similar in both contrast-enhanced (CE) and non-CE lesions. (B) A significantly higher content of RAM-11–positive cells (macrophages) were detected in CE than in non-CE lesions. (C) In contrast, the amount of α-actin–positive cells (smooth muscle cells) was similar in both type of lesions.

From the imaging view point, an interesting observation was the higher number of neovessels in the arterial wall (adventitia and plaque) in CE-positive lesions compared with the CE-negative lesions (Fig. 3A). A more detailed analysis clearly showed that the difference was mostly due to the increased intraplaque angiogenesis (plaque vasorum) (Fig. 3B); the number of adventitial neovessels was similar among the different lesions (Fig. 3C). These findings are in line with the selected CEU imaging criteria at visual examination defining as CE-positive those lesions with signal contrast agent enhancement within the lesion, and as CE-negative those lesions with no signal within the plaque or confined to the plaque adventitial side.

(A) The total number of vessels was significantly higher in contrast-enhanced (CE) than in non-CE atherosclerotic lesions. The increased contrast enhancement was related to increased intraplaque neovascularization as demonstrated by the higher intraplaque (B), but not adventitial (C), neovessel density in CE- versus non-CE plaques.

A positive correlation between the number of intraplaque neovessels and contrast signal enhancement of the lesions was noted (Fig. 6). Figures 6A through 6C show the strong contrast enhancement of a CE plaque and the corresponding pathological specimen showing high density of neovessels at immunohistochemistry (Figs. 6E and 6F).

Before (A) and after (B) contrast-administration. (C) Contrast enhancement quantification by digital subtraction imaging. Green area depicts contrast enhancement of the representative contrast-enhanced (CE)–positive lesion. (D) Positive strong correlation between the total number of neovessels and the signal enhancement in fibrofatty lesions is shown. At 25× magnification (E) and 200× magnification (F), a histological section of the imaged atherosclerotic plaque stained for lectin (neovessels) is shown. Asterisk indicates the corresponding area in the ultrasound images and in the histological section from the same atherosclerotic lesion. Arrows in E and F indicate the corresponding luminal surface of the plaques in the ultrasound images and in the histological section from the same animal.

Discussion

We have described the feasibility of CEU imaging for the noninvasive, in vivo visualization of neovascularization in atherosclerotic lesions in a rabbit model of advanced atherosclerosis. The findings were corroborated by histological analyses of the corresponding lesions. The increased plaque angiogenesis associated with macrophage-rich lesions was clearly identified by rapid contrast enhancement of the lesions. On the other hand, more fibrotic lesions with lower macrophage content did not show a similar enhancement. The specificity of the contrast agent used in the study was demonstrated by the lack of lesion enhancement after saline administration in atherosclerotic animals. Similarly, no contrast enhancement in nonatherosclerotic control rabbits after contrast injection was observed.

It is important to outline that the greater neovessel density at histology was observed in CE-positive lesions than in CE-negative lesions. These findings are in agreement with previous data showing a good correlation between vasa vasorum and the degree of contrast agent enhancement in human atherosclerotic lesions (15). However, the atherosclerotic lesions used in that study were highly stenotic because they had to meet the criteria for endarterectomy and therefore did not include lesions with <50% stenosis (20,21). In fact, the high-risk lesions may not always be highly stenotic (22). The use of an animal model of atherosclerosis overrides this limitation, allowing the inclusion of lesions with a greater degree of stenosis. The present study classified lesions not only according to their contrast enhancement but also the histological cellular composition and neovessel density of the imaged lesions.

Interestingly, CEU imaging was able to differentiate plaques according to their vascularization despite their similar sizes. Our results also show a higher macrophage density in CE-positive than in CE-negative plaques, whereas vascular smooth muscle cell density was similar in both groups. Moreover, CE-negative lesions were characterized by a greater fibrotic component than CE-positive plaques. These observations are concordant with those of a previous report showing the relationship between neovascularization assessed by contrast agent enhancement and plaque composition, irrespective of plaque size (15).

Similar results were found when categorizing the atherosclerotic lesions according to their echogenicity. Class III lesions, which showed a significantly greater increase in contrast enhancement compared with class II plaques, demonstrated a similar plaque area but a greater content of macrophages compared with class II lesions. Class III lesions were also characterized by increased neovessel density than class II plaques. These results further confirm that in predominantly echogenic lesions, the increased macrophage density was associated with higher neovessel content, a finding in line with previous observations showing that increased density of intraplaque neovessels is associated with plaque instability (23–25) as well as increased macrophage infiltration (7,15,26). The possibility of directly imaging increased neovascularization may significantly improve plaque characterization over traditional ultrasound. In fact, despite our findings showing that predominantly echogenic lesions also show increased neovascularization and macrophage density, CEU imaging may provide a unique opportunity to monitor the serial progressive pathophysiological developments of intraplaque neovessels (27). Moreover, future application using targeted microbubbles targeting markers of endothelial activation/dysfunction or damage or angiogenesis receptors (28–31) might help to elucidate the mechanism underlying neoangiogenesis in atherosclerosis.

Another novelty of this study concerns the significant association seen between CE-positive lesions and plaque vasorum but not adventitial neovessels compared with CE-negative lesions. These findings suggest that contrast enhancement specifically reflects intraplaque rather than adventitial vascularization. Different results have been reported by others (13,16) describing increased adventitial and intraplaque neovascularization detected by CEU imaging in human carotid atherosclerotic lesions. However, those observations were not corroborated by the systematic histological evaluation of the imaged lesions.

A likely explanation for our findings showing no correlation between contrast enhancement and adventitial neovessels could be related to the speed of penetration of the contrast. Indeed, after contrast injection, the lumen was opacified for about 1 min, not allowing any earlier evaluation of contrast enhancement. Therefore, it is possible that adventitial neovessel enhancement, mostly occurring in this early phase, is actually either underestimated or undetectable. Moreover, a careful adjustment of imaging settings is required to detect contrast agent microbubbles penetrating plaque tissue. After tissue signal suppression, however, the plaque appears dark, allowing easy detection of the microbubbles, the adventitia remains bright, and this could actually limit the detection ability of the technique. Finally, although the experimental animal model used in the present study is characterized by advanced human-like atherosclerotic lesions, as measured by not only lipid content but also by fibrotic areas (17), we cannot rule out some discrepancy compared with human atherosclerotic lesions.

Conclusions

The results of the present study support CEU imaging as a feasible technique for the noninvasive assessment of intraplaque neovascularization. Several findings suggest that intraplaque neovascularization is associated with plaque progression and vulnerability (9). Therefore, the availability of a noninvasive technique to assess lesion neovascularization could be of great importance to identify highly vascularized lesions. It has been suggested that inhibition of plaque neovessels could be a potential pharmacological target to promote atherosclerotic lesion stabilization. Therefore, the noninvasive assessment of plaque neovascularization could be of great importance in detecting the presence of highly vascularized lesions and possibly to establish the intensity of antiatherosclerotic treatments (i.e., lipid-lowering approaches).

Acknowledgments

The authors thank Josè Rodriguez, Boris Cortez, and Noemi Escalera for the proper conduct of the experimental work.

Appendix

For a supplemental video, please see the online version of this article.

Footnotes

The authors have reported that they have no relationships to disclose.